The present invention relates to a protein factor involved in angiogenesis in humans and falls in the field of genetic engineering.
The process of angiogenesis, in which endothelial cells existing in the inner wall of blood vessels of animals generate new blood vessels, is triggered by transduction of a specific signal. A variety of substances are reportedly involved in this signal transduction. The most notable substance among them is the vascular endothelial growth factor (VEGF). VEGF is a protein factor which was isolated and purified, and can increase the proliferation of endothelial cells and the permeability of blood vessels (Senger, D. R. et al., Science 219: 983-985 (1983); Ferrara, N. and Henzel, W. J. Biochem. Biophys. Res. Commun. 161: 851-858 (1989)). It has been reported that the human VEGF gene contains eight exons and produces four subtypes consisting of 121, 165, 189, or 206 amino acid residues, depending on the difference in splicing, which causes different secretionpatterns (Houck, K. A. et al., Mol. Endocrinol. 5:1806-1814 (1991)). It has also been reported that there is a VEGF-specific receptor, flt-1, and that the binding of VEGF to flt-1 is important for the signal transduction (Vries, C. D. et al., Science 255: 989-991 (1992)).
Placental growth factor (PlGF) and platelet-derived growth factor (PDGF) have thus far been isolated and are factors related to VEGF. These factors are found to promote proliferation activities of vascular endothelial cells (Maglione, D. et al., Proc. Natl. Acad. Sci. USA 88: 9267-9271 (1991); Betsholtz, C. et al., Nature 320: 695-699(1986)). In addition, VEGF-B (Olofsson, B. et al., Proc. Natl. Acad. Sci. USA 93: 2576-2581 (1996)) and VEGF-C (Lee, J. et al., Proc. Natl. Acad. Sci. USA 93: 1988-1992 (1996); Joukov, V. et al., EMBO J. 15, 290-299 (1996)) have recently been isolated.
These factors appear to constitute a family, and this may contain additional unknown factors.
It has been suggested that VEGF is involved in not only vascular formation at the developmental stage but also in the pathological neovascularization associated with diabetes, rheumatoid arthritis, retinopathy, and the growth of solid tumors. Furthermore, in addition to its vascular endothelial cell growth-promoting effects listed above, VEGF""s ability to increase vascular permeability was suggested to be involved in the edema formation resulting from various causes. Also, these VEGF family factors may act on not only the blood vessels but also the blood cells and the lymphatic vessels. They may thus play a role in the differentiation and proliferation of blood cells and the formation of lymphatic vessels. Consequently, the VEGF family factors are presently drawing extraordinary attention for developing useful, novel drugs.
An objective of the present invention is to isolate a novel protein belonging to the VEGF family and a gene encoding the protein. We searched for genes having homology to VEGF-C, which is a recently cloned VEGF family gene, against Expressed Sequence Tags (EST) and Sequence Tagged Sites (STS) in the GenBank database. As a result, we found an EST that was assumed to have homology to the C-terminal portion of VEGF-C. We then designed primers based on the sequence, and amplified and isolated the corresponding cDNA using the 51 RACE method and the 3xe2x80x2 RACE method. The nucleotide sequence of the isolated cDNA was determined, and the deduced amino acid sequence therefrom revealed that the amino acid sequence had significant homology to that of VEGF-C. Based on the homology, we have assumed that the isolated human clone is a fourth member of the VEGF family (hereinafter designated as VEGF-D). We have also succeeded in expressing the protein encoded by the isolated human VEGF-D gene in E. coli cells, and have also purified and isolated it. Furthermore, we have succeeded in isolating the mouse and rat VEGF-D genes using the isolated human VEGF-D gene. In particular, the present invention relates to a novel protein belonging to the VEGF family and a gene encoding the protein. More specifically it relates to
(1) A protein shown by SEQ ID NO: 1 or having the amino acid sequence derived therefrom in which one or more amino acids are substituted, deleted, or added;
(2) A protein encoded by a DNA that hybridizes with the DNA shown by SEQ ID NO. 2;
(3) A DNA encoding the protein of (1);
(4) A DNA hybridizing with the DNA shown by SEQ ID NO: 2;
(5) A vector containing the DNA of (3) or (4);
(6) A transformant carrying the vector of (5);
(7) A method of producing the protein of (1) or (2), which comprises culturing the transformant of (6);
(8) An antibody binding to the protein of (1) or (2);
(9) A method of screening a compound binding to the protein of (1) or (2), which comprises a step of detecting the activity of the protein of (1) or (2) to bind to a test sample; and
(10) A compound binding to the protein of (1) or (2), wherein said compound has been isolated by the method of (9).
The protein of the present invention (VEGF-D) has significant homology to VEGF-C and can be considered to be a fourth factor of the VEGF family. Since the major function of VEGF is vascular formation at the developmental stage and VEGF is considered to be involved in the pathological neovascularization associated with diabetes, rheumatoid arthritis, retinopathy, and the growth of solid tumors, the protein of the present invention is thought to have similar functions.
A person skilled in the art could prepare functionally equivalent proteins through modifying VEGF-D of the present invention by adding, deleting, or substituting one or more of the amino acids of VEGF-D shown by SEQ ID NO: 1 using known methods. Modifications of the protein can also occur naturally in addition to the artificial modifications described above. These modified proteins are also included in the present invention. Known methods for adding, deleting, or substituting amino acids include the overlap extension polymerase chain reaction (OE-PCR) method (Gene, 1989, 77 (1): 51).
The DNA encoding VEGF-D of the present invention, shown by SEQ ID NO: 2, is useful for isolating DNAs encoding the proteins having similar functions to VEGF-D in other organisms. For example, a person skilled in the art could routinely isolate homologs of human VEGF-D of the present invention from other organisms by allowing the DNA shown by SEQ ID NO: 2, or part thereof, as a probe, to hybridize with the DNA derived from other organisms. The DNA that hybridizes with the DNA shown by SEQ ID NO: 2 is also included in the present invention. The other organisms include mice, rats, and rabbits.
The DNA encoding a protein that is functionally equivalent to VEGF-D usually has high homology to the DNA shown by SEQ ID NO: 2. The high homology used herein means at least 70% or higher, more preferably 80% or higher, and still more preferably 90% or higher of sequence homology.
An example of the hybridization conditions for isolating the DNA having high homology will be given below. Prehybridization is performed in ExpressHyb Solution at 68xc2x0 C. for 30 minutes. The probe labeled with a radioisotope is denatured at 95xc2x0 C. to 100xc2x0 C. for 2 to 5 minutes and rapidly chilled on ice. The probe is added to a new ExpressHyb Solution. The blot is transferred to the solution containing the probe and allowed to hybridize under a temperature gradient of 68xc2x0 C. to 55xc2x0 C. for 2 hours. The blot is washed four times, for 10 minute each, with a 2xc3x97SSC solution containing 0.05% SDS at room temperature. The blot is then washed with a 0.1xc3x97SSC solution containing 0.1% SDS at 45xc2x0 C. for 3 minutes. The blot is subjected to autoradiography.
An example of the hybridization conditions for isolating the DNA having very high homology will be given below. Prehybridization is performed in ExpressHyb Solution at 68xc2x0 C. for 30 minutes. The probe labeled with a radioisotope is denatured at 95xc2x0 C. to 100xc2x0 C. for 2 to 5 minutes and rapidly chilled on ice. The probe is added into a new ExpressHyb Solution. The blot is transferred into the solution containing the probe, and allowed to hybridize at 68xc2x0 C. for 1 hour. The blot was washed four times, for 10 minute each, with a 2xc3x97SSC solution containing 0.05% SDS at room temperature. The blot was then washed with a 0.1xc3x97SSC solution containing 0.1% SDS at 50xc2x0 C. for 40 minutes, during which the solution was replaced once. The blot was then subjected to autoradiography.
Note that the hybridization condition can vary depending on the length of the probe (whether it is an oligomer or a probe with more than several hundred bases), the labeling method (whether the probe is radioisotopically labeled or non-radioisotopically labeled) and the type of the target gene to be cloned. A person skilled in the art would properly select the suitable hybridization conditions. In the present invention, it is especially desirable that the condition does not allow the probe to hybridize with the DNA encoding VEGF-C.
The DNA of the present invention is also used to produce VEGF-D of the present invention as a recombinant protein. Specifically, the recombinant protein can be produced in large quantity by incorporating the DNA encoding VEGF-D (for example, the DNA shown by SEQ ID NO: 2) into a suitable expression vector, introducing the resulting vector into a host, and culturing the transformant to allow the recombinant protein to be expressed.
The vector to be used for producing the recombinant protein is not particularly restricted. However, vectors such as pGEMEX-1 (Promega) or pEF-BOS (Nucleic Acids Res. 1990, 18(17): p.5322) are preferable. Suitable examples of the host into which the vector is introduced include E. coli cells, CHO cells, and COS cells.
The VEGF-D protein expressed by the transformant can be purified by suitably combining purification treatments such as solubilization with a homogenizer or a sonicator, extraction by various buffers, solubilization or precipitation by acid or alkali, extraction or precipitation with organic solvents, salting out by ammonium sulfate and other agents, dialysis, ultrafiltration using membrane filters, gel filtration, ion exchange chromatography, reversed-phase chromatography, counter-current distribution chromatography, high-performance liquid chromatography, isoelectric focusing, gel electrophoresis, or affinity chromatography in which antibodies or receptors are immobilized.
Once the recombinant protein is obtained, antibodies against it can be prepared using known methods. The known methods include preparing polyclonal antibodies by immunizing rabbits, sheep, or other animals with the purified protein, and preparing monoclonal antibodies from the antibody-producing cells of immunized mice or rats. These antibodies will make it possible to quantify VEGF. Although the antibodies thus obtained can be used as they are, it will be more effective to use the humanized antibodies to reduce the immunogenicity. The methods of humanizing the antibodies include the CDR graft method and the method of directly producing a human antibody. In the CDR Graft method, the antibody gene is cloned from the monoclonal antibody-producing cells and its antigenic determinant portion is transplanted into an existing human antibody. In the method of directly producing a human antibody, a mouse whose immune system has been replaced by the human immune system is immunized, similar to ordinary monoclonal antibodies. The VEGF-D protein or its antibody thus obtained can be administered into the body by subcutaneous injection or a similar method.
A person skilled in the art could screen compounds that bind to the protein of the present invention by known methods.
For example, such compounds can be obtained by making a cDNA library on a phage vector (such as xcexgt11 and ZAP) from the cells expected to express the protein that binds to the protein of the present invention (such as lung, small intestine, and heart cells of mammals), expressing the cDNAs on LB-agarose, fixing the expressed proteins onto a filter, preparing the purified protein of the present invention as a biotin-labeled or a fusion protein with the GST protein, and reacting this protein with the above filter. The desired compounds could then be detected by west western blotting using streptavidin or an anti-GST antibody (Skolnik, E. Y., Margolis, B., Mohammadi, M., Lowenstein, E., Fischer, R., Drepps, A., ullrich, A., and Schlessinger, J. (1991) Cloning of P13 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases, Cell 65: 83-90). Another method comprises the following steps. First, express the protein of the present invention fused with the SRF binding domain or the GAL4 binding domain in yeast cells. Second, prepare a cDNA library which expresses cDNAs fused with the transcription activation domain of VP16 or GAL4 from the cells expected to express a protein that binds to the protein of the present invention. Third, introduce the cDNA into the above yeast cells. Fourth, isolate the library-derived cDNA from the positive clones. Finally, introduce the isolated cDNA into E. coli to allow it to be expressed. (When a protein that binds to the protein of the present invention is expressed in yeast cells, the reporter gene is activated and the positive clone can be detected.) This method can be performed using the two-hybrid system (MATCHMAKER Two-Hybrid System, Mammalian MATCHMAKER Two-Hybrid Assay Kit, or MATCHMAKER One-Hybrid System (all by Clontech) orthe HybriZAP Two-Hybrid Vector System (Stratagene) (Dalton, S. and Treisman, R. (1992) Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element, Cell 68: 597-612). Alternatively, the binding proteins can be screened by preparing a cDNA library from the cells expected to express a substance, such as a receptor, which binds to the protein of the present invention (for example, vascular endothelial cells, bone marrow cells, or lymph duct cells), introducing it into such cells as COS, detecting the binding of the protein of the present invention by itself or labeled with a radioisotope or a fluorescence, and cloning proteins that bind to the protein of the present invention (Yamasaki, K., Taga, T., Hirata, Y., Yawata, H., Kawanishi, Y., Seed, B., Taniguchi, T., Hirano, T., and Kishimoto, T. (1988) Cloning and expression of human interleukin-6 (BSF-2/IFN beta2) receptor, Science 241: 825-828, Fukunaga, R., Ishizaka-Ikeda, E., Seto, Y., and Nagata, S. (1990) Expression cloning of a receptor for murine granulocyte colony-stimulating factor, Cell 61: 341-350). Still another method comprises applying the culture supernatant or the cellular extract of the cells expected to express a protein that binds to the protein of the present invention onto an affinity column to which the protein of the present invention has been immobilized, and purifying the proteins specifically bound to the column. In addition, a DNA encoding the protein that binds to the protein of the present invention can be obtained by determining the amino acid sequence of the binding protein, synthesizing oligonucleotides based on the sequence, and screening a cDNA library with the oligonucleotides as probes.
Furthermore, compounds that bind to the protein of the present invention can be screened by contacting compounds, a natural substance bank, or a random phage peptide display library with the immobilized protein of the present invention and detecting the molecules bound to the protein. These compounds can also be screened by high throughput screening utilizing combinatorial chemistry technology (Wrighton, N. C., Farrell, F. X., Chang, R., Kashyap, A. K., Barbone, F. P., Mulcahy, L. S., Johnson, D. L., Barrett, R. W., Jolliffe, L. K., and Dower, W. J., Small peptides as potent mimetics of the protein hormone erythropoietin, Science (United States) Jul. 26, 1996, 273: 458-464, Verdine, G. L., The combinatorial chemistry of nature, Nature (England) Nov. 7, 1996, 384: 11-13, Hogan, J. C. Jr. Directed combinatorial chemistry, Nature (England) Nov. 7, 1996, 384: 17-19).
VEGF-D of the present invention may be used for gene therapy by introducing the VEGF-D gene into the body of the patient with the VEGF-D deficiency, or expressing the gene in the body. An anti-sense DNA of the VEGF-D gene may also be used to inhibit the expression of the gene itself, thereby suppressing the pathological neovascularization.
Among the many available methods to introduce the VEGF-D gene or its antisense DNA into the body, the retrovirus method, the liposome method, the cationic liposome method, and the adenovirus method are preferable.
In order to express these genes in the body, the genes can be incorporated into a suitable vector and introduced into the body by the retrovirus method, the liposome method, the cationic liposome method, or the adenovirus method. Although the vectors to be used are not particularly limited, such vectors as pAdexlcw and pZIPneo are preferable.
The present invention may also be applied for diagnosing disorders caused by abnormalities of the VEGF-D gene, for example, by PCR to detect an abnormality of the nucleotide sequence of the VEGF-D gene.
Furthermore, according to the present invention, the VEGF-D protein or its agonists can be used to heal wounds, promote collateral vessel formation, and aid hematopoiesis by the hematopoietic stem cells, by taking advantage of the angiogenic effect of the VEGF-D protein. The antibodies against the VEGF-D protein or its antagonists can be used as the therapeutic agents for pathological neovascularization, lymphatic dysplasia, dyshematopoiesis, or edemas arising from various causes. The anti-VEGF-D antibodies can be used for diagnosing diseases resulting from abnormal production of VEGF-D by quantifying VEGF-D.